Cytotoxic T-lymphocytes (CTLs) are a critical component of effective human immune responses to tumors or viral infections. Cytotoxic T-lymphocytes destroy neoplastic cells or virus infected cells through recognition of antigenic peptides presented by MHC class I molecules on the surface of the affected target cells. These antigenic peptides are degradation products of foreign proteins present in the cytosol of the affected cell, which are processed and presented to CTLs through the endogenous MHC class I processing pathway.
Although the recognition of a foreign protein in the context of the MHC class I molecule may be sufficient for the recognition and destruction of affected target cells by CTLS, the induction of antigen-specific CTLs from T-lymphocyte precursors requires additional signals. Specialized antigen presenting cells (APCs) can provide both the antigen-MHC class I ligand and the accessory signals required in the induction phase of CTL-mediated immunity. General properties of APCs include MHC class I and class II expression, expression of various adhesion molecules important for APC-lymphocyte interaction, and expression of costimulatory molecules such as CD80 and CD86. Examples of APCs include macrophages and dendritic cells (including cutaneous epidermal Langerhans cells, dermal dendritic cells, and dendritic cells resident in lymph nodes and spleen).
Attempts to induce antigen-specific CTL responses in vivo by immunization with killed tumor cells, killed virus-infected cells, or component proteins have generally been unsuccessful, presumably because proteins in the extracellular fluids cannot enter the cytosol and access the MHC class I presentation pathway.
Genetic immunization possess several attractive features. Several in vivo gene transfer methods result in transgene expression, including retroviral or adenoviral mediated gene transfer, and direct injection of naked DNA (for a review, see Krishnaw, et al., 1995, Nature Med. 1: 521–522 and Pardoll, et al., 1995, Immunity 3:165–169.)
Williams, et al. (1991, Proc. Natl. Acad. Sci. USA 88: 2726–2730) showed the expression of the protein luciferase in intact epidermal cells following biolistic (biobalistic) delivery of the firefly luciferase gene. CTL responses were not addressed in these studies, nor were specific host cells targeted to generate cell-mediated immune responses.
Tang, et al. (1992, Nature 356: 152–154) utilized a biolistic (biobalistic) device to produce a humoral response to a foreign protein. A gene encoding hGH under control of either the CMV promoter or the β-actin promoter was delivered to the epidermal tissue of mice. Anti-hGH antibodies were detected in mice in response to this immunization procedure. Tang, et al. does not disclose genetic immunization targeting the cell-mediated immune pathway. Direct targeting of APC cells for genetic immunization is not disclosed or suggested by Tang, et al.
Fynan, et al. (1993, Proc. Natl. Acad. Sci. USA 90: 11478–11482) confirmed the findings of Tang, et al. by using a plasmid DNA construct encoding an influenza virus hemagglutinin glycoprotein. Fynan, et al. compared humoral responses generated by gene gun delivery of DNA coated gold beads to the epidermis with other mechanisms and found that the use of a biolistic (biobalistic) device 1) resulted in 95% protection to a lethal influenza challenge, 2) was the most efficient route for DNA immunization, proving to be substantially more effective than mucosal, intramuscular, or intravenous administration, and, 3) required 250 to 2500 times less DNA than saline inoculations. Direct targeting of APC cells for genetic immunization is not disclosed or suggested by Fynan, et al. CTL-mediated immunity is not addressed.
Liu and colleagues (Montgomery et al., 1993, DNA Cell Biol. 12:777–783; Ulmer et al., 1993, Science. 259:1745–1749; Donnelly et al., 1995, Nature Medicine 1:583–587.) have demonstrated that untargeted, nonspecific intramuscular injection of naked DNA induces antigen-specific CTL responses to viral proteins and protective immunity to viral challenge. The studies do not disclose targeting of genetic material to APCs for genetic immunization.
Sun, et al. (1995, Proc. Natl. Acad. Sci. USA 92: 2889–2893) utilized a biolistic (biobalistic) device to produce an anti-tumor response in mice. The authors delivered a plasmid construct expressing IL-6 directly to a tumor site in mice. Expression of IL-6 afforded a form of cytokine gene therapy nonspecifically directed at the tumor. Antigen-specific immunity to tumors was not claimed. Direct targeting of APC cells for genetic immunization is not disclosed or suggested by Sun, et al.
Kundig et al. (1995, Science. 268:1343–1346) demonstrate that protein antigen localization to the lymphoid organs is critical for the induction of antigen-specific CTL responses in vivo. Genetic immunization is not addressed.
Kovacsovics-Bankowski and Rock (1995, Science 267: 243–246) demonstrate a phagosome-to-cytosol pathway for protein antigens not normally presented through the MHC class I endogenous pathway. The authors speculate that proteins in particulate form internalized within phagosomes are in fact able to enter the cytosolic pathway for MHC class I presentation. The capacity of functionally intact genetic material to enter the cytosol through a phagosome-to-cytosol pathway is not addressed.
Falo, et al. (1995, Nature Med. 1: 649–653) offer in vivo support for phagosome-to-cytosol pathway by showing that delivery of particulate protein antigen directly into animals results in antigen-specific CTL mediated tumor immunity in mice. They demonstrate that proteins injected directly into animals in vivo can specifically enter the phagosome-cytosol pathway of APCs if administered in particulate form. No details are forwarded regarding genetic immunization procedures. The capacity of in vivo administered genetic material to enter the cytosol of APCs, or other cell types, functionally intact through this pathway is not addressed.
Pardoll and Beckerleg (1995, Immunity 3:165–169) have recently reviewed the immunology of naked DNA vaccines. They emphasize the importance of additional studies to define the currently unknown mechanism of DNA immunization. Specifically, they conclude that “it will be important to dissect the mechanisms by which it (naked DNA) activates immune responses. It is only through these studies that intelligent modifications can be introduced to maximize both qualitatively and quantitatively its ultimate potency.”
Despite the efforts documented in the above reference material, there remains a need to develop a genetic immunization protocol which specifically targets cell types within the host to stimulate antigen-specific CTL mediated immunity and in turn promote direct destruction of specific neoplastic or virally infected cells within the host. The present invention both addresses and meets this need.